The present invention relates to a method for increasing sensitivity and specificity of nucleic acid synthesis by reducing nonspecific nucleic acid synthesis occurring at ambient temperature. The invention also relates to novel nucleic acids which have high affinity to polymerases. The methods and compositions of the present invention can be used in DNA sequencing, amplification reactions, nucleic acid synthesis and cDNA synthesis.
The invention also relates to nucleic acids and compositions which are capable of inhibiting or preventing nucleic acid synthesis, sequencing, amplification and cDNA synthesis, for example, by binding one or more polypeptides with polymerase activity. In addition, the materials and methods of the present invention may be used as therapeutics to inhibit the replication of organisms that rely upon a reverse transcriptase activity for completion of their life cycle, such as retroviruses. The invention also relates to vectors and host cells comprising such nucleic acid molecules. The invention also concerns kits comprising the compositions or nucleic acids of the invention.
DNA polymerases synthesize the formation of DNA molecules which are complementary to a DNA template. Upon hybridization of a primer to the single-stranded DNA template, polymerases synthesize DNA in the 5xe2x80x2 to 3xe2x80x2 direction, successively adding nucleotides to the 3xe2x80x2-hydroxyl group of the growing strand. Thus, in the presence of deoxyribonucleoside triphosphates (dNTPs) and a primer, a new DNA molecule, complementary to the single stranded DNA template, can be synthesized.
Both mesophilic and thermophilic DNA polymerases are used to synthesize nucleic acids. Using thermostable rather than mesophilic polymerases is preferable since the higher annealing temperatures used with thermostable polymerases result in less non-specific DNA amplification from extension of mis-annealed primers. Even with thermostable polymerases, however, some primer sequences and certain experimental conditions can result in the synthesis of a significant amount of non-specific DNA products. These non-specific products can reduce the sensitivity of polymerase-based assays and can require extensive optimization for each primer set. In addition, this problem is intensified when polymerases having a high level of activity at ambient temperature are employed (for example, DNA polymerase from Thermotoga neapolitana).
In examining the structure and physiology of an organism, tissue or cell, it is often desirable to determine its genetic content. The genetic framework of an organism is encoded in the double-stranded sequence of nucleotide bases in the deoxyribonucleic acid (DNA) which is contained in the somatic and germ cells of the organism. The genetic content of a particular segment of DNA, or gene, is only manifested upon production of the protein and RNA which the gene encodes. In order to produce a protein, a complementary copy of one strand of the DNA double helix (the xe2x80x9ccodingxe2x80x9d strand) is produced by polymerase enzymes, resulting in a specific sequence of ribonucleic acid (RNA). This particular type of RNA, since it contains the genetic message from the DNA for production of a protein, is called messenger RNA (mRNA).
Within a given cell, tissue or organism, there exist many mRNA species, each encoding a separate and specific protein. This fact provides a powerful tool to investigators interested in studying genetic expression in a tissue or cell. mRNA molecules may be isolated and further manipulated by various molecular biological techniques, thereby allowing the elucidation of the full functional genetic content of a cell, tissue or organism.
A common approach to the study of gene expression is the production of complementary DNA (cDNA) clones. In this technique, the mRNA molecules from an organism are isolated from an extract of the cells or tissues of the organism. This isolation often employs chromatography matrices, such as cellulose or agarose, to which oligomers of thymidine (T) have been complexed. Since the 3xe2x80x2 termini on most eukaryotic mRNA molecules contain a string of Adenosine (A) bases, and since A binds to T, the mRNA molecules can be rapidly purified from other molecules and substances in the tissue or cell extract. From these purified mRNA molecules, cDNA copies may be made using the enzyme reverse transcriptase (RT) or DNA polymerases having RT activity, which results in the production of single-stranded cDNA molecules. The single-stranded cDNAs may then be converted into a complete double-stranded DNA copy (i.e., a double-stranded cDNA) of the original mRNA (and thus of the original double-stranded DNA sequences, encoding this mRNA, contained in the genome of the organism) by the action of a DNA polymerase. The protein-specific double-stranded cDNAs can then be inserted into a vector, which is then introduced into a host bacterial, yeast, animal or plant cell, a process referred to as transformation or transfection. The host cells are then grown in culture media, resulting in a population of host cells containing (or in many cases, expressing) the gene of interest or portions of the gene of interest.
This entire process, from isolation of mRNA to insertion of the cDNA into a vector (e.g., plasmid, viral vector, cosmid, etc.) to growth of host cell populations containing the isolated gene or gene portions, is termed xe2x80x9ccDNA cloning.xe2x80x9d If cDNAs are prepared from a number of different mRNAs, the resulting set of cDNAs is called a xe2x80x9ccDNA library,xe2x80x9d an appropriate term since the set of cDNAs represents a xe2x80x9cpopulationxe2x80x9d of genes or portions of genes comprising the functional genetic information present in the source cell, tissue or organism.
Synthesis of a cDNA molecule initiates at or near the 3xe2x80x2 termini of the mRNA molecules and proceeds in the 5xe2x80x2-to-3xe2x80x2 direction successively adding nucleotides to the growing strand. Priming of the cDNA synthesis at the 3xe2x80x2-termini at the poly A tail using an oligo (dT) primer ensures that the 3xe2x80x2 message of the mRNAs will be represented in the cDNA molecules produced. The ability to increase sensitivity and specificity during cDNA synthesis provides more representative cDNA libraries and may increase the likelihood of the cDNA library having full-length cDNA molecules (e.g., full-length genes). Such advances would greatly improve the probability of finding full-length genes of interest.
In addition to their importance for research purposes, reverse transcriptase enzymes play a critical role in the life cycle of many important pathogenic viruses, in particular, the human immunodeficiency viruses (HIV). In order to complete its life cycle, HIV and other similar viruses must use a reserve transcriptase enzyme to convert the viral RNA genome into DNA for integration into the host""s genomic material. Since this step is critical to the viral life cycle and host cells do not have any similar requirement for reverse transcriptase activity, the reverse transcriptase enzyme has been intensively studied as a chemotherapeutic target. In general, the bulk of therapeutic reagents directed at the reverse transcriptase enzyme have been nucleotide analogues, for example AZT. Other therapeutic modalities using oligonucleotide-based reagents, e.g., anti-sense oligonucleotides and ribozymes, have been used to inhibit viral replication, however, these reagents are not targeted specifically against reverse transcriptase activity, instead of being targeted against the nucleic acid of the viral genome. See, for example, Goodchild, et al., xe2x80x9cInhibition of human immunodeficiency virus replication by antisense oligodeoxynucleotides,xe2x80x9d Proc. Natl. Acad. Sci. USA 85:5507-5511 (1988), Matsukara, et al., xe2x80x9cRegulation of viral expression of human immunodeficiency virus in vitro by an antisense phosphorothioate oligodeoxynucleotide against rev (art/trs) in chronically infected cells,xe2x80x9d Proc. Natl. Acad Sci. USA 86:4244-4248 (1989), Rossi, et al., xe2x80x9cRibozymes as Anti-HIV-1 Therapeutic Agents: Principles, Applications, and Problems,xe2x80x9d Aids Research and Human Retroviruses 8:183:189 (1992), Goodchild, xe2x80x9cEnhancement of ribozyme catalytic activity by a contiguous oligodeoxynucleotide (facilitator) and by 2xe2x80x2-O-methylation,xe2x80x9d Nucleic Acids Research 20:4607-4612 (1992) and Kinchington, et al., xe2x80x9cA comparison of gag, pol and rev antisense oligodeoxynucleotides as inhibitors of HIV-1,xe2x80x9d Antiviral Research 17:53-62 (1992) which are specifically incorporated herein by reference. Oligonucleotides that have been blocked at the 3xe2x80x2-end to prevent their elongation by reverse transcriptase have also been considered as inhibitors (see, for example, Austermann, et al., xe2x80x9cInhibition of human immunodeficiency virus type 1 reverse transcriptase by 3xe2x80x2-blocked oligonucleotidesxe2x80x9d Biochemical Pharmacology 43(12):2581-2589 (1992). Each of the above cited references is specifically incorporated herein in its entirety.
Oligonucleotides have been investigated for anti-HIV activity. For example, Idriss, et al. (1994), Journal of Enzyme Inhibition 8(2)97-112, disclose DNA oligonucleotides in a hairpin structure as inhibitors of HIV RT activity while Kuwasaki, et al., (1996) Biochemical and Biophysical Research Communications 228:623-631 disclose anti-sense hairpin oligonucleotides containing a mixture of deoxy and 2xe2x80x2-methoxy-nucleotides with anti-HIV activity.
Notwithstanding these and other efforts to modulate the activity of polymerases, there remains a need in the art for materials and methods to prevent the undesirable activity of the polymerases while permitting the synthesis of nucleic acids by the polymerase when such synthesis is desired. These and other needs are met by the present invention.
The present invention provides materials and methods for inhibiting, reducing, substantially reducing or eliminating nucleic acid synthesis under certain conditions (preferably at ambient temperatures and/or within a cell) while permitting synthesis when such synthesis is desired.
In a preferred aspect, the invention relates to methods for the prevention or inhibition of nucleic acid synthesis during reaction set up (e.g., in vitro) and preferably before optimum reaction conditions for nucleic acid synthesis are achieved. Such inhibition of synthesis at sub-optimum conditions or during reaction set up prevents or reduces non-specific nucleic acid synthesis. Once reaction set up is complete and the optimum conditions are reached, nucleic acid synthesis can be initiated.
In another aspect, the present invention relates to a method of inhibiting a polymerase enzyme within a cell (e.g., in vivo) by introducing into the cell an oligonucleotide or inhibitor of the invention, preferably said oligonucleotide comprises a 5xe2x80x2- and a 3xe2x80x2-portion, wherein the said 3xe2x80x2-portion comprises one or more deoxyribonucleotides or derivatives thereof and said 5xe2x80x2-portion comprises one or more ribonucleotides or derivatives thereof and wherein all or a portion of said 3xe2x80x2-portion is capable of base pairing to all or a portion of said 5xe2x80x2-portion and incubating said cell under conditions causing the inhibition of the polymerase. In some embodiments, the 5xe2x80x2-portion of the oligonucleotide which comprises ribonucleotides forms a 5xe2x80x2-overhang. In another aspect, the oligonucleotide is in the form of a hairpin and preferably the stem of the hairpin comprises a series of contiguous ribonucleotides based paired or hybridized with a series of continguous deoxyribonucleotides. In some embodiments the polymerase is a reverse transcriptase and may preferably be an HIV reverse transcriptase.
In another aspect, the present invention provides a method of inhibiting replication of a virus, by providing a virus, said virus comprising a reverse transcriptase and requiring activity of the reverse transcriptase for replication and contacting said reverse transcriptase with an oligonucleotide or inhibitor of the invention that inhibits activity of said reverse transcriptase thereby inhibiting replication of said virus. In some embodiments, the oligonucleotide comprises a 5xe2x80x2- and a 3xe2x80x2-portion, wherein said 3xe2x80x2-portion comprises one or more deoxyribonucleotides or derivatives thereof and said 5xe2x80x2-portion comprises one or more ribonucleotides or derivatives thereof and wherein all or a portion of said 3xe2x80x2-portion is capable of base pairing to all or a portion of said 5xe2x80x2-portion. In some embodiments, the 5xe2x80x2-portion of the oligonucleotide which comprises ribonucleotides forms a 5xe2x80x2-overhang. In another aspect, the oligonucleotide is in the form of a hairpin and preferably the stem of the hairpin comprises a series of contiguous ribonucleotides base paired or hybridized with a series of contiguous deoxyribonucleotides. In some embodiments, the virus is an HIV. In some embodiments, contacting comprises introducing said oligonucleotide into a cell.
More specifically, the invention relates to controlling nucleic acid synthesis by introducing an inhibitory nucleic acid or oligonucleotide which binds to or interacts with the polypeptide with polymerase activity (e.g., DNA polymerases, reverse transcriptases, etc.). Accordingly, such inhibitory nucleic acids or oligonucleotide can bind the polymerase and interfere with nucleic acid synthesis by preventing binding or interaction of the polymerase or reverse transcriptase with the primer/template. Preferably, such inhibitory nucleic acid molecules are double stranded molecules although any form of nucleic acid molecule may be used as long as the molecule can bind or interact with the polymerization enzyme of interest. Such molecules may be DNA, RNA, DNA/RNA hybrids, double stranded DNA, double stranded RNA and DNA/RNA double stranded molecules. Derivative nucleic acid molecules may also be used such as Protein Nucleic Acids (PNAs), linked nucleic acids (LNA, available form Proligo, Boulder Colo.) and nucleic acid molecules comprising modified nucleotides. Moreover, the nucleic acid molecules may be in any form or topology such as linear, circular, supercoiled, double stranded with one or more single stranded portions, hairpin structure, or complexed with other molecules such as peptides or proteins and the like. Such inhibitory nucleic acids preferably include double-stranded nucleic acid molecules (which may comprise one or more internal, 5xe2x80x2 and/or 3xe2x80x2 single stranded portions), or single stranded nucleic acid molecules capable of folding into a double stranded form, i.e. forming one or more hairpin-loops, such that at least one double stranded portion of the nucleic acid molecule is capable of binding to a polypeptide with polymerase activity. In one aspect, the nucleic acid molecules used in the invention bind the polypeptide having polymerase activity (e.g., DNA polymerase, reverse transcriptase, etc.) with high affinity. Once the polymerase or reverse transcriptase is complexed with the inhibitory nucleic acid, it is unavailable for annealing to the primer/template substrate, resulting in reduced, substantially reduced, or no polymerase or reverse transcriptase activity. In some embodiments, the oligonucleotides of the present invention may comprise a 5xe2x80x2- and a 3xe2x80x2-portion, wherein said 3xe2x80x2-portion comprises one or more deoxyribonucleotides or derivatives thereof and said 5xe2x80x2-portion comprises one or more ribonucleotides or derivatives thereof and wherein all or a portion of said 3xe2x80x2-portion is capable of base pairing to all or a portion of said 5xe2x80x2-portion. In some embodiments, the oligonucleotides of the invention may comprise a 5xe2x80x2-portion, wherein said 5xe2x80x2-portion comprising ribonucleotides forms a 5xe2x80x2-overhang. In some embodiments, an oligonucleotide of the invention may comprise one or more modifications so as to be non-extendable. In some embodiments, this modification may be to the 3xe2x80x2-most nucleotide. In some embodiments, the modification is phosphorylation of the 3xe2x80x2-most nucleotide at the 3xe2x80x2-hydroxyl. An oligonucleotide of the present invention may comprise one or more modifications so as to be resistant to digestion or degradation by, for example, one or more nucleases. In some embodiments, this modification may be the incorporation of one or more phophorothioate moieties. In some embodiments, the modification may comprise alkylation of one or more hydroxl groups.
Thus, the inhibitory nucleic acid is preferably introduced into the reaction mixture where it competitively binds to or interacts with the polymerase, thereby inhibiting synthesis by the polymerase under particular reaction conditions. Thus, interaction or binding of the inhibitor and polymerase preferably results in the formation of an inhibitor/polymerase complex.
The inhibition of polymerase activity or nucleic acid synthesis by the nucleic acids of the invention is preferably reduced, substantially reduced, inhibited, or eliminated so that nucleic acid synthesis may proceed when reaction conditions are changed, for example, when the temperature is raised. In a preferred aspect, the changed conditions affect the ability of the inhibitory nucleic acids to interact with the polymerase causing release of the polymerase and/or denaturation or inactivation of the inhibitory nucleic acids making the polymerase available thus allowing nucleic acid synthesis to proceed. In one aspect, the inhibitory nucleic acids and the primer/template substrate competitively interact with the polymerase to prevent synthesis. Under the changed conditions, the competitive interaction is reduced such that nucleic acid synthesis occurs. In another aspect, the changed conditions cause the double stranded inhibitory nucleic acid molecule(s) (including hairpins) to denature or melt such that single stranded molecules are formed which do not substantially bind or interact with the polymerase. In another aspect, a second change in conditions (i.e., temperature is lowered to, for example, ambient temperatures) allows the inhibitor nucleic acid molecules of the invention to reactivate or again inhibit nucleic acid synthesis. That is, the inhibitors may again interact or bind with the polymerase or reverse transcriptase under the changed conditions. For example, the changed conditions may allow the inhibitor to form double stranded molecules which effectively enhances its binding or interacting capacity with the polymerase or reverse transcriptases. Thus, in accordance with the invention, the inhibitors may be reused or recycleable during synthesis reactions (single or multiple) which may require multiple adjustment or changes in reaction conditions (i.e., temperature changes), without the need to add additional inhibitor.
The invention therefore relates to a method for synthesizing one or more nucleic acid molecules, comprising (a) mixing one or more nucleic acid templates (which may be a DNA molecule such as a cDNA molecule, or an RNA molecule such as an mRNA molecule) with one or more primers, and one or more inhibitory nucleic acids or compositions of the present invention capable of binding or interacting with an enzyme having polymerase activity, and (b) incubating the mixture in the presence of one or more enzymes having nucleic acid polymerase activity (e.g., DNA polymerases or reverse transcriptases) under conditions sufficient to synthesize one or more first nucleic acid molecules complementary to all or a portion of the templates. Alternatively, the method may comprise mixing one or more inhibitor nucleic acids with one or more polymerases and incubating such mixtures under conditions sufficient to synthesize one or more nucleic acid molecules. Such conditions may involve the use of one or more nucleotides and one or more nucleic acid synthesis buffers. Such methods of the invention may optionally comprise one or more additional steps, such as incubating the synthesized first nucleic acid molecule under conditions sufficient to make a second nucleic acid molecule complementary to all or a portion of the first nucleic acid molecule. These additional steps may also comprise the use of the inhibitory nucleic acid molecules of the invention. The invention also relates to nucleic acid molecules synthesized by these methods.
In a related aspect, the nucleic acid synthesis method may comprise (a) mixing one or more polymerases with one or more of the inhibitory nucleic acid molecules of the invention, and (b) incubating such mixture under conditions sufficient to inactivate or substantially inhibit or reduce polymerase activity of such polymerases. In another aspect, such incubation is under conditions sufficient to inhibit or prevent such nucleic acid synthesis.
The invention also relates to a method for amplifying one or more nucleic acid molecules, comprising (a) mixing one or more nucleic acid templates with one or more primers, and one or more inhibitory nucleic acid molecules or compositions of the present invention capable of binding or interacting with an enzyme having polymerase activity and (b) incubating the mixture in the presence of one or more enzymes having nucleic acid polymerase activity (e.g., DNA polymerases) under conditions sufficient to amplify one or more nucleic acid molecules complementary to all or a portion of the templates. More specifically, the invention relates to a method of amplifying a DNA molecule comprising: (a) providing a first and second primer, wherein said first primer is complementary to a sequence within or at or near the 3xe2x80x2-termini of the first strand of said DNA molecule and said second primer is complementary to a sequence within or at or near the 3xe2x80x2-termini of the second strand of said DNA molecule, and one or more inhibitory nucleic acids or compositions of the invention (e.g., a nucleic acid having affinity for an enzyme with polymerase activity); (b) hybridizing said first primer to said first strand and said second primer to said second strand; (c) incubating the mixture under conditions such that a third DNA molecule complementary to all or a portion of said first strand and a fourth DNA molecule complementary to all or a portion of said second strand are synthesized; (d) denaturing said first and third strand, and said second and fourth strands; and (e) repeating steps (a) to (c) or (d) one or more times. Such conditions may include incubation in the presence of one or more polymerases, one or more nucleotides and/or one or more buffering salts. The invention also relates to nucleic acid molecules amplified by these methods.
In a related aspect, the nucleic acid amplification method may comprise (a) mixing one or more polymerases with one or more of the inhibitory nucleic acid molecules of the invention, and (b) incubating such mixture under conditions sufficient to inactivate or substantially inhibit or reduce polymerase activity of such polymerases. In another aspect, such incubation is under conditions sufficient to inhibit or prevent such nucleic acid amplification.
The invention also relates to methods for sequencing a nucleic acid molecule comprising (a) mixing a nucleic acid molecule to be sequenced with one or more primers, one or more of the inhibitory nucleic acids or compositions of the invention, one or more nucleotides and one or more terminating agents to form a mixture; (b) incubating the mixture under conditions sufficient to synthesize a population of molecules complementary to all or a portion of the molecule to be sequenced; and (c) separating the population to determine the nucleotide sequences of all or a portion of the molecule to be sequenced. The invention more specifically relates to a method of sequencing a nucleic acid molecule, comprising: (a) providing an inhibitory nucleic acid or composition of the present invention (to which an enzyme with polymerase activity as affinity), one or more nucleotides, and one or more terminating agents; (b) hybridizing a primer to a first nucleic acid molecule; (c) incubating the mixture of step (b) under conditions sufficient to synthesize a random population of nucleic acid molecules complementary to said first nucleic acid molecule, wherein said synthesized molecules are shorter in length than said first molecule and wherein said synthesized molecules comprise a terminator nucleotide at their 3xe2x80x2 termini; and (d) separating said synthesized molecules by size so that at least a part of the nucleotide sequences of said first nucleic acid molecule can be determined. Such terminator nucleotides include dideoxyribonucleoside thiphophates such as ddNTP, ddATP, ddGTP, ddITP or ddCTP. Such conditions may include incubation in the presence of one or more polymerases and/or buffering salts.
In a related aspect, the nucleic acid sequencing method may comprise (a) mixing one or more polymerases with one or more of the inhibitory nucleic acid molecules of the invention, and (b) incubating such mixture under conditions sufficient to inactivate or substantially inhibit polymerase activity of such polymerases. In another aspect, such incubation is under conditions sufficient to inhibit or prevent such nucleic acid sequencing.
The invention also relates to the inhibitory nucleic acids of the invention and to compositions comprising the inhibitory nucleic acids of the invention, to vectors (which may be expression vectors) comprising these nucleic acid molecules, and to host cells comprising these nucleic acid molecules or vectors. Compositions of the invention may also include those compositions made for carrying out the methods of the invention or produced while carrying out such methods. The invention also relates to pharmaceutical compositions. Such compositions may comprise one or more of the inhibitory nucleic acid molecules or oligonucleotides of the invention and at least one other component selected from the group consisting of one or more nucleotides, one or more polymerases (e.g., thermophilic or mesophilic DNA polymerases and/or reverse transcriptases), one or more suitable buffers or buffer salts, one or more primers, one or more terminating agents, one or more viruses, one or more cells, and one or more amplified or synthesized nucleic acid molecules produced by the methods of the invention. The invention also relates to methods of producing an inhibitory nucleic acid comprising culturing the above-described host cells under conditions favoring the production of the nucleic acid by the host cells, and isolating the nucleic acid. The invention also relates to nucleic acid produced by synthetic methods. Such inhibitory nucleic acid molecules of the invention may also be made by standard chemical synthesis techniques.
In a related aspect, the present invention provides materials and methods for the in vivo inhibition of polymerase activity. In some embodiments, the present invention provides for the introduction of the inhibitory oligonucleotides of the present invention into an organism thereby inhibiting a polymerase present within the organism. In some embodiments, the polymerase may be a reverse transcriptase, preferably a viral reverse transcriptase. In some embodiments, the present invention provides a method for the inhibition of a viral reverse transcriptase comprising contacting a cell or virus expressing a viral reverse transcriptase with an inhibitory oligonucleotide under conditions causing the oligonucleotide to inhibit the reverse transcriptiase. Preferably, the oligonucleotide is contacted with the cell under conditions sufficient to have the oligonucleotide taken up by the cell by well known techniques. In some embodiments, the present invention provides a method of inhibiting the growth of a virus, comprising contacting a cell infected with a virus that requires reverse transcriptase activity to complete its life cycle with an inhibitory oligonucleotide under conditions causing the oligonucleotide to be taken up by the cell and causing the reverse transcriptase to be inhibited thereby inhibiting the growth of the virus. In some embodiments, the present invention provides a method of treating an organism or subject infected with a virus that requires reverse transcriptase activity to complete its life cycle comprising contacting an infected cell of the organism or subject with a composition comprising an inhibitory oligonucleotide under conditions causing the oligonucleotide to be taken up by the cell and causing the reverse transcriptase to be inhibited thereby treating the organism.
The invention also relates to kits for use in synthesis, sequencing and amplification of nucleic acid molecules, comprising one or more containers containing one or more of the inhibitory nucleic acids or compositions of the invention. These kits of the invention may optionally comprise one or more additional components selected from the group consisting of one or more nucleotides, one or more polymerases (e.g., thermophilic or mesophilic DNA polymerases and/or reverse transcriptases), one or more suitable buffers, one or more primers and one or more terminating agents (such as one or more dideoxynucleotides). The invention also relates to kits for inhibiting viral replication or kits for treating viral infections comprising the inhibitory nucleic acids of the invention. Such kits may also comprise instructions or protocols for carrying out the methods of the invention.
Other preferred embodiments of the present invention will be apparent to one of ordinary skill in light of the following drawings and description of the invention, and of the claims.